The research in this proposal deals with the characterization of genetic and biochemical mechanisms responsible for the initiation of replication and the stable maintenance of the broad host-range plasmid RK2 in bacteria. In addition to its medical importance in specifying resistance to the commonly used antibiotics tetracycline, kanamycin and ampicillin in a wide range of bacteria, plasmid RK2 serves as a model system for understanding the initiation of DNA replication and the distribution, possibly by a partitioning mechanism, of DNA elements to daughter cells upon cell division. RK2 encodes a replication initiation protein (TrfA) and a replication origin sequence that has as its main feature eight 17 base pair direct repeats (iterons). In addition, a 3.2 kilobase segment of the plasmid contains two operons, designated parCBA and parDE, that stably maintain the plasmid in Escherichia coli and other Gram-negative bacteria. The parDE operon encodes a post-segregational killing mechanism that corrects for loss of the plasmid in a growing population of bacteria while the parCBA operon provides effective plasmid stabilization by an unknown mechanism that may involve plasmid partitioning. The proposed studies of the initiation of RK2 replication are directed at understanding biochemical events at the RK2 origin of replication including the contribution of the TrfA protein and the host DnaA protein to the formation of an open complex and the possible interaction of the TrfA protein with the DnaBC protein complex and other host replication proteins from E. coli and other bacteria. These studies will use both wild-type and mutant forms of the TrfA protein and a variety of in vitro techniques including a replication system utilizing purified proteins from E. coli. Furthermore, the role of coupled complexes between RK2 origins of replication and the TrfA protein will be further assessed both with regard to structure and role in regulation of plasmid copy-number. The analysis of the RK2 stabilization region will emphasize elucidating the mechanism of plasmid stable maintenance encoded by the parCBA operon and the biochemical roles of the ParC, B and A proteins in this process. These studies are likely to contribute to our understanding of the genetic and biochemical basis of broad host-range replication and stable maintenance properties of medically important bacterial plasmids.